"In vitro" correction of the severe factor V deficiency-related coagulopathy by a novel plasma-derived factor V concentrate.

INTRODUCTION: Severe congenital factor V (FV) deficiency is a rare bleeding disorder characterized by very low/undetectable levels of FV. Fresh frozen plasma is the standard treatment for bleeding manifestations. Recently, a novel plasma-derived FV concentrate has been developed. AIM: To evaluate the "in vitro" ability of the novel FV concentrate to normalize clotting times and generate normal amount of thrombin in plasma collected from patients with severe FV deficiency. METHODS: Prothrombin time (PT), activated partial thromboplastin time (aPTT), FV activity and antigen levels and thrombin generation were measured pre- and postspiking of plasma samples of 10 patients with increasing doses of FV concentrate (from 0 to 100 IU/dL). RESULTS: Prothrombin time and activated partial thromboplastin time ratios as well as all thrombin generation parameters were fully corrected by the addition of FV concentrate at a final concentration of 25 IU/dL. However, the addition of FV at a concentration of 1-3 IU/dL was already sufficient to correct peak height and endogenous thrombin potential (but not lag time and time to peak) after activation with 5 pmol/L tissue factor. FV activity and antigen levels showed a linear response to supplementation with the novel FV concentrate. CONCLUSION: The novel plasma-derived FV concentrate was effective to correct "in vitro" severe FV deficiency in patients. The optimal FV concentration to fully normalize both global clotting times and thrombin generation parameters using the novel plasma-derived FV concentrate was 25 IU/dL.

Characterization of an apparently synonymous F5 mutation causing aberrant splicing and factor V deficiency.

Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. We investigated a patient with severe FV deficiency (FV:C < 3%) and moderate bleeding symptoms. Thrombin generation experiments showed residual FV expression in the patient's plasma, which was quantified as 0.7 ± 0.3% by a sensitive prothrombinase-based assay. F5 gene sequencing identified a novel missense mutation in exon 4 (c.578G>C, p.Cys193Ser), predicting the abolition of a conserved disulphide bridge, and an apparently synonymous variant in exon 8 (c.1281C>G). The observation that half of the patient's F5 mRNA lacked the last 18 nucleotides of exon 8 prompted us to re-evaluate the c.1281C>G variant for its possible effects on splicing. Bioinformatics sequence analysis predicted that this transversion would activate a cryptic donor splice site and abolish an exonic splicing enhancer. Characterization in a F5 minigene model confirmed that the c.1281C>G variant was responsible for the patient's splicing defect, which could be partially corrected by a mutation-specific morpholino antisense oligonucleotide. The aberrantly spliced F5 mRNA, whose stability was similar to that of the normal mRNA, encoded a putative FV mutant lacking amino acids 427-432. Expression in COS-1 cells indicated that the mutant protein is poorly secreted and not functional. In conclusion, the c.1281C>G mutation, which was predicted to be translationally silent and hence neutral, causes FV deficiency by impairing pre-mRNA splicing. This finding underscores the importance of cDNA analysis for the correct assessment of exonic mutations.

Genetic modulation of the FV(Leiden)/normal FV ratio and risk of venous thrombosis in factor V Leiden heterozygotes.

BACKGROUND AND OBJECTIVES: The factor (F)V Leiden mutation causes activated protein C (APC) resistance by decreasing the susceptibility of FVa to APC-mediated inactivation and by impairing the APC-cofactor activity of FV in FVIIIa inactivation. However, APC resistance and the risk of venous thromboembolism (VTE) vary widely among FV Leiden heterozygotes. Common F5 genetic variation probably contributes to this variability. PATIENTS/METHODS: APC resistance was determined in 250 FV Leiden heterozygotes and 133 normal relatives using the prothrombinase-based assay, which specifically measures the susceptibility of plasma FVa to APC. The effects of 12 F5 single-nucleotide polymorphisms (SNPs) on the normalized APC sensitivity ratio (nAPCsr) and on FV levels were determined by multiple regression analysis. RESULTS: In FV Leiden heterozygotes,VTE risk increased with increasing nAPCsr, reaching an odds ratio (OR) of 9.9 (95% confidence interval [CI] 1.2­80.5) in the highest nAPCsr quartile. The minor alleles of several F5 SNPs, including 327 A/G (Q51Q), 409 G/C (D79H), 2663 A/G(K830R, T2 haplotype), 6533 T/C (M2120T) and 6755 A/G (D2194G, R2 haplotype), increased the nAPCsr in FV Leiden heterozygotes, but not in their normal relatives. Most of these effects could be attributed to a shift in the FV(Leiden)/normal FV ratio. Four FV Leiden heterozygotes with extremely high nAPCsr turned out to be pseudo-homozygotes, i.e. they carried a deleterious mutation on the non-Leiden allele. CONCLUSIONS: In FV Leiden heterozygotes, the prothrombinase-based nAPCsr is a marker of VTE risk and is modulated by common F5 SNPs that affect the FV(Leiden)/normal FV ratio in plasma.

Whole blood rotation thromboelastometry (ROTEM(®)) in nine severe factor V deficient patients and evaluation of the role of intraplatelets factor V.

Severe factor V (FV) deficiency (parahaemophilia) is a rare congenital hemorrhagic disorder characterized by very low or undetectable plasma FV levels and bleeding phenotype ranging from mild to severe. We evaluated whole blood (WB) rotation thromboelastometry (ROTEM) in parahaemophilia patients and the contribution of intraplatelets FV, if any, to clot formation. Standard ROTEM(®) assays were performed in WB from nine parahaemophilia patients and 50 healthy controls. In addition, platelets poor plasma from one parahaemophilia patient (PPP-Pt) or normal subjects (PPP-N) was reconstituted with washed platelets obtained either from one patient with parahaemophilia (Plts-Pt) or normal subjects (Plts-N) and ROTEM assays were performed in platelets rich plasma (PRP) samples. There was a prolongation of the WB clotting time (CT) in all assays in patients as compared with controls. However, maximum clot firmness (MCF) was similar in patients and controls. ROTEM in PPP-Pt showed both a prolongation of CT and a reduction of MCF as compared with PPP-N. The addition of either Plts-Pt or Plts-N to PPP-Pt resulted in similar increase in MCF and a decrease of CT which was more evident for PPP-Pt + Plts-N than PPP-Pt + Plts-Pt. In contrast, the addition of Plts-Pt or Plts-N to PPP-N had superimposable effects on both CT and MCF. In parahaemophilia patients, WB ROTEM(®) presents mainly with prolongation of CT and no relevant effect on MCF. Residual intraplatelets FV in parahaemophilia contributes significantly to thrombin generation as shown in artificially reconstituted PRP models.